Metastatic colorectal cancer (mCRC) is the second leading cause of cancer-related death in the US. Roughly half of mCRCs harbor a mutation in BRAF or KRAS, associated with worse prognosis and fewer treatment options. Results of clinical trials targeting components of the signaling cascades containing BRAF and KRAS have been underwhelming. These findings signify that mCRC is not driven by a unique pathway or dominated by a single oncogene. The objective of this proposal is to test the hypothesis that a critical set of upregulated, parallel pathways function as cooperative dependencies, driving therapeutic resistance in BRAF and KRAS mutated mCRC. New resources and technologies are required to accomplish this objective. Thus, the proposed research will pair patient-representative specimens, including tumor samples and patient- derived xenograft (PDX) models established by co-PI, Dr. Atreya, with a novel high-throughput kinase-activity mapping (HT-KAM) platform developed by co-PI, Dr. Copp. HT-KAM uses peptide libraries as combinatorial sensors to directly measure the activity of kinase enzymes and reveal actionable vulnerabilities of kinase pathways. Aims 1 and 2 will focus on BRAF(V600E) mutated mCRC. The starting materials for Aim 1 are BRAF mutated mCRC PDX tumors treated with regimens received by corresponding patients on clinical trials, or a BRAF inhibitor combined with agents targeting orthogonal modes of action, found by surveying CRC cell lines with a pilot version of HT-KAM. Aim 1 will identify the conserved kinase-dependent pathways of BRAF mutated PDX tumors. Kinase signatures will be established via a vastly expanded version of HT-KAM, and computationally integrated into hierarchies of functional networks using the PhosphoAtlas blueprint of the phospho-reactome built by Dr. Copp. This will reveal which pathways and which specific dependencies are most conserved. Aim 2 will test new combinatorial strategies to treat BRAF mutated mCRC, identified from pilot studies and the kinome profiles of PDX tumors. Two- to four-drug strategic possibilities, directed at distinct pathways, will be prioritized from candidate targets. Treatment hypotheses will be tested in cell lines and synergy analysis will be performed. The most promising combinations will then be tested in BRAF mutated mCRC PDX models. The regimens with the greatest efficacy will be optimized in anticipation of translation back to patients. Aim 3 will focus on KRAS(G12) mutated mCRC. Preliminary data predicts that the functional susceptibilities driving BRAF and KRAS mutated mCRC will differ. KRAS(G12) mutated mCRC offers the opportunity to directly evaluate surgical specimens, and to develop corresponding PDX models. The same iterative workflow as described for BRAF mutated mCRC will be applied to discover and target the cooperative dependencies of KRAS(G12) mutated mCRC. The long-term goals of this project are to are: 1) to improve the survival of patients via therapeutic strategies specifically tailored to BRAF or KRAS mCRC; and 2) to establish a transformative discovery pipeline that is generalizable to any tumor type.